CN114149496B

CN114149496B - Tumor angiogenesis regulatory protein and application thereof

Info

Publication number: CN114149496B
Application number: CN202111462706.6A
Authority: CN
Inventors: 鹿文葆
Original assignee: Institute of Microcirculation of CAMS
Current assignee: Institute of Microcirculation of CAMS
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2023-08-22
Anticipated expiration: 2041-12-03
Also published as: CN114149496A

Abstract

The application provides a tumor angiogenesis regulating protein, which comprises Arid5a and/or Arid5b. The tumor angiogenesis regulatory protein has important theoretical value for the mechanism research of tumor angiogenesis, tumor growth and metastasis, and has important application value for tumor treatment.

Description

Tumor angiogenesis regulatory protein and application thereof

Technical Field

The application relates to the fields of oncology, molecular genetics and molecular biology, in particular to a tumor angiogenesis regulating protein and application thereof.

Background

Tumor angiogenesis is a key step in the further growth and metastasis of solid tumors (Weinberg RA, 2008). However, the molecules that control tumor angiogenesis and their mechanisms remain unclear. Tumor cells activate vascular endothelial cells by secreting and releasing some angiogenesis-related factors, which in turn induce the production of a rich vascular network within the tumor tissue (humimann Y, et al, 2008), which will promote further growth of the tumor and distant organ metastasis (villar C et al, 2017). Thus, a series of angiogenic factors secreted by tumor cells become a tie between tumor cells and vascular endothelial cells (Weis SM et al 2011), which determine whether tumor angiogenesis is on or off. Typically, the opening of most tumor angiogenesis is accomplished by either increasing the expression of a pro-angiogenic factor gene or by decreasing the expression of an anti-angiogenic factor gene. Whether it balances or not will determine whether angiogenesis is induced or inhibited (Otrock ZK et al, 2007). While recent advances have expanded our understanding of the mechanism by which tumor cells induce angiogenesis, the mechanism formed by the development of posttranscriptional mechanisms in tumor cells that controls the balance of angiogenic gene expression, and in particular the role and mechanism by which RNA-binding proteins play in controlling this balance, remains unclear.

Arid5a (AT-rich interactive domain-containing protein a) is a double stranded RNA encoded by the arod 5a geneBinding proteins, belonging to the ARID (AT-rich interaction domain) family members. Arid5a and Arid5b are homologous proteins, and both Arid5a and Arid5b have tumor-inhibiting effects, which are exemplified below by Arid5 a. Studies have shown that the deletion of Arid5a upregulates the inflammatory factor IL6 and triggers a severe autoinflammatory response. Arid5a ^-/- Mice are effective against bleomycin (bleomycin) induced lung injury. Arid5a may be involved in rheumatoid arthritis by binding its N-terminal region to RORγt (Retinoic acid receptor-related orphan nuclear receptor γt) and inhibiting Th17 cell differentiation. In addition, arid5a can modulate primary CD4 by specifically stabilizing Stat3 mRNA ⁺ T cell differentiation. It was also found that Arid5a stabilized T-bet mRNA and exacerbated IFN-gamma mediated infective shock. Notably, the cytoplasmic localization of Arid5a is critical for its mRNA stabilizing function. These studies indicate that Arid5a has an immune function modulating function. In addition, arid5a can be used as a cell quiescence (cell quiescence) marker molecule and a potential therapeutic target for metabolic diseases. Taken together, these reports suggest that Arid5a plays an important role in disease development and cellular progression. However, it is currently unclear as to the role of Arid5a in tumor progression, especially the signaling pathway regulated by Arid5a in human tumor cells and its target genes. Therefore, the elucidation of the regulatory role of Arid5a in tumor-induced angiogenesis and the mechanism thereof has very important application prospects for future targeted tumor angiogenesis treatment of cancers.

Disclosure of Invention

Aiming at the problems in the prior art, the application provides a tumor angiogenesis regulatory protein, which has important theoretical value for the mechanism research of tumor angiogenesis, tumor growth and metastasis and has important application value for tumor treatment.

In a first aspect, the application provides a tumor angiogenesis regulating protein, which comprises Arid5a and/or Arid5b, wherein the amino acid sequence of Arid5a is shown as SEQ ID NO. 1.

In a second aspect, as shown in SEQ ID NO.2, the present application proposes a gene encoding the tumor angiogenesis regulating protein.

In a third aspect, the present application provides a protein truncate, which is the Arid functional region of the Arid5a protein of claim 1, and/or which is the Arid functional region of the Arid5b protein of claim 1.

In a fourth aspect, the application provides a gene encoding said Arid5a protein truncate or Arid5b protein truncate.

In a fifth aspect, the present application provides an application of the tumor angiogenesis regulating protein or the protein truncated body in preparing a tumor therapeutic drug; the functions of the medicine are as follows (a 1) and/or (a 2) and/or (a 3): (a 1) treating a tumor; (a 2) inhibiting tumor growth and/or metastasis; (a 3) inhibiting tumor angiogenesis.

In a sixth aspect, the present application provides a biomaterial prepared from the gene.

In a seventh aspect, the application provides an application of the biological material prepared by the gene in preparing a tumor therapeutic drug; the functions of the medicine are as follows (a 1) and/or (a 2) and/or (a 3): (a 1) treating a tumor; (a 2) inhibiting tumor growth and/or metastasis; (a 3) inhibiting tumor angiogenesis.

In an eighth aspect, the present application provides a substance that can up-regulate the abundance of the Arid5a and/or Arid5b proteins in an organism prepared from the tumor angiogenesis regulating protein.

In a ninth aspect, the present application provides an application of the substance capable of up-regulating the abundance of the Arid5a and/or Arid5b protein in an organism in preparing a tumor therapeutic drug; the functions of the medicine are as follows (a 1) and/or (a 2) and/or (a 3): (a 1) treating a tumor; (a 2) inhibiting tumor growth and/or metastasis; (a 3) inhibiting tumor angiogenesis.

In a tenth aspect, the tumor angiogenesis regulating protein and/or the gene are used as targets in the development of antitumor drugs.

Drawings

FIG. 1 is a graph showing in vitro and in vivo inhibition of tumor angiogenesis by Arid5a, wherein both MDA-MB-231/Arid5a-GFP cells and MDA-MB-468/Arid5a-GFP cells efficiently express an Arid5a-GFP fusion protein (FIG. 1A); overexpression of the Arid5a gene significantly inhibited vascular cell migration and tube formation induced by tumor cell conditioned medium (FIG. 1B, FIG. 1C); overexpression of the Arid5a gene inhibited angiogenesis in tumor tissues (fig. 1D and 1E);

FIG. 2 shows that Arid5 a-targeted 3' UTRs selectively promote mRNAs expression of tumor angiogenesis inhibitor genes. After overexpression of the Arid5a gene, the anti-angiogenic genes mRNAs were up-regulated and the pro-angiogenic genes were down-regulated (FIG. 2A, FIG. 2B, FIG. 2C); analysis of Gene Ontology (Gene Ontology) data showed that the 'angiogensis' term was significantly enriched in the Arid5a regulated Gene (fig. 2D); the Arid5a protein can target mRNAs that bind to anti-angiogenic genes, but do not bind to pro-angiogenic genes (FIG. 2E); the Arid5a protein can target the 3' UTR binding to the anti-angiogenic gene up-regulate its mRNAs expression (FIGS. 2F and 2G);

FIG. 3 shows that the ARID domain of Arid5a is responsible for stabilizing mRNAs of angiogenesis inhibitor genes. Detection of angiogenesis inhibitor gene mRNAs half-life it was found that overexpression of the Arid5a gene significantly increased their half-life (fig. 3A, 3B, 3C and 3D); schematic structural diagrams of Arid5a functional regions and preparation strategies of different mutants (FIG. 3E); immunoblotting identified the expression of the different mutants of Arid5a (fig. 3F); after overexpression of the T1 or T3 truncate genes, the angiogenesis inhibiting gene mRNAs are up-regulated (fig. 3G); t1 truncations or T3 truncations may target 3' UTRs that bind angiogenesis inhibiting genes to stabilize their mRNAs (FIG. 3H); overexpression of the T1 truncate gene or the T3 truncate gene inhibits angiogenesis induced by tumor cell conditioned medium (FIG. 3I);

FIG. 4 shows that in vivo overexpression of the Arid5a gene inhibits tumor growth and metastasis, wherein the recombinant adenovirus expressing the Arid5a gene can significantly inhibit tumor growth and metastasis in nude mice (FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D), and the recombinant adenovirus expressing the Arid5a gene can significantly inhibit angiogenesis in tumor tissues (FIG. 4E).

Detailed Description

The application aims at providing novel uses of Arid5a, arid5b proteins and Arid5a and/or Arid5b genes, and more particularly relates to novel uses of Arid5a, arid5b proteins and Arid5a, arid5b genes in preparing tumor therapeutic drugs and novel uses of targets in anti-tumor angiogenesis drug development.

The application firstly protects the application of the Arid5a and/or Arid5b protein in preparing the anti-tumor angiogenesis therapeutic drugs.

The application also protects application of the Arid5a and/or Arid5b genes or biological materials with the Arid5a and/or Arid5b genes in preparing tumor therapeutic drugs.

The application also protects the application of the substances capable of up-regulating the abundance of the Arid5a and/or Arid5b proteins in organisms in preparing tumor therapeutic drugs. The substance capable of up-regulating the abundance of the Arid5a and/or Arid5b protein in the organism may be the Arid5a and/or Arid5b protein itself, or may be other proteins or gene sequences located upstream of the Arid5a and/or Arid5b protein in the organism that promote the production of the Arid5a and/or Arid5b protein, or may be other proteins located downstream of the Arid5a and/or Arid5b protein in the organism that reduce the degradation of the Arid5a and/or Arid5b protein, or may be compounds or other small molecules that promote the increase in the level of the Arid5a and/or Arid5b protein in the organism.

The application also protects the application of the substances capable of up-regulating the abundance of the Arid5a and/or Arid5b genes in organisms in preparing tumor therapeutic drugs. The substance capable of up-regulating the abundance of the Arid5a and/or Arid5b genes in the organism may be the Arid5a and/or Arid5b genes themselves, other proteins or nucleic acid molecules located upstream of the Arid5a and/or Arid5b genes in the organism that can promote the expression of the Arid5a and/or Arid5b genes, other proteins or nucleic acid molecules located downstream of the Arid5a and/or Arid5b genes in the organism that can reduce the degradation of the Arid5a and/or Arid5b genes, or compounds or other small molecules that can promote the expression of the Arid5a and/or Arid5b genes in the organism.

The application also protects application of the Arid5a and/or Arid5b protein or the Arid5a and/or Arid5b gene as a target in the research and development of anti-tumor angiogenesis drugs. Arid5a and/or Arid5b proteins may be upregulated as targets. The substance that can up-regulate the Arid5a and/or Arid5b protein in the organism may be the Arid5a and/or Arid5b protein itself, other proteins that can promote the production of the Arid5a and/or Arid5b protein upstream of the Arid5a and/or Arid5b protein in the organism, other proteins that can reduce the degradation of the Arid5a and/or Arid5b protein downstream of the Arid5a and/or Arid5b protein in the organism, or compounds or other small molecules that can promote the increase in the level of the Arid5a and/or Arid5b protein in the organism. The Arid5a and/or Arid5b gene may specifically be upregulated as a target. The substance that up-regulates the Arid5a and/or Arid5b gene in the organism may be the Arid5a and/or Arid5b gene itself, or may be other proteins or nucleic acid molecules that promote expression of the Arid5a and/or Arid5b gene in the organism upstream of the Arid5a and/or Arid5b gene, or may be other proteins or nucleic acid molecules that reduce degradation of the Arid5a and/or Arid5b gene downstream of the Arid5a and/or Arid5b gene in the organism, or may be compounds or other small molecules that promote expression of the Arid5a and/or Arid5b gene in the organism.

The application also protects an Arid5a and/or Arid5b protein truncate, i.e. an Arid functional region of the Arid5a and/or Arid5b protein.

The ARID functional region of the Arid5a and/or Arid5b protein is amino acid residues 55-148 of the Arid5a and/or Arid5b protein.

Genes encoding the arod 5a and/or arod 5b protein truncations are also within the scope of the application.

The application also protects application of the Arid5a and/or Arid5b protein truncations in preparing tumor treatment medicines.

The application also protects the application of the gene for encoding the Arid5a and/or Arid5b protein truncated body or the biological material with the gene for encoding the Arid5a protein truncated body in preparing tumor therapeutic drugs.

The application also protects application of the Arid5a and/or Arid5b protein truncations or genes encoding the Arid5a and/or Arid5b protein truncations as target objects in the research and development of antitumor drugs. The Arid5a protein truncate may specifically be an Arid5a and/or Arid5b protein truncate that is upregulated as a target. The substance that can up-regulate the Arid5a and/or Arid5b protein truncations in the organism can be the Arid5a and/or Arid5b protein truncations themselves, as well as other proteins, peptide fragments, compounds or other small molecules that can promote increased levels of Arid5a and/or Arid5b protein truncations in the organism. The gene encoding the Arid5a and/or Arid5b protein truncations may specifically be upregulated as a target for the gene encoding the Arid5a and/or Arid5b protein truncations. The substance that can up-regulate the gene encoding the Arid5a and/or Arid5b protein truncations in the organism can be the gene itself encoding the Arid5a protein truncations, or can be other proteins, polypeptides, nucleic acid molecules, compounds or other small molecules that promote expression of the gene encoding the Arid5a and/or Arid5b protein truncations.

The function of any of the above drugs is (a 1) and/or (a 2) and/or (a 3) as follows: (a 1) treating a tumor; (a 2) inhibiting tumor growth and/or metastasis; (a 3) inhibiting tumor angiogenesis.

Any of the above mentioned Arid5a and/or Arid5b proteins may be human Arid5a and/or Arid5b proteins.

Any of the above mentioned Arid5a and/or Arid5b proteins may specifically be (b 1), (b 2), (b 3) or (b 4) as follows:

(b1) A protein shown in SEQ ID NO.1 in the sequence table;

(b2) A fusion protein obtained by ligating a tag to the amino terminus or the carboxyl terminus of (b 1);

(b3) A protein having any one of the functions (a 1) to (a 3) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of (b 1);

(b4) A protein which is derived from a human and has 98% or more of identity with (b 1) and has any one of functions (a 1) to (a 3).

The labels are specifically shown in table 1.

TABLE 1 sequence of tags

The Arid5a and/or Arid5b protein may also be a homologous protein having any of the functions (a 1) to (a 3) in other species. Such other species include, but are not limited to, mice, rats, rabbits, dogs, monkeys, chimpanzees, apes, cattle, sheep, pigs, horses, sheep, goats, cats, and the like.

The Arid5a and/or Arid5b genes are genes encoding the Arid5a and/or Arid5b proteins.

Any of the above mentioned Arid5a and/or Arid5b genes may specifically be (c 1) or (c 2) or (c 3) as follows:

(c1) A DNA molecule with a coding region shown as SEQ ID NO.2 in the sequence table;

(c2) A DNA molecule derived from a human and having more than 95% identity to (c 1) and encoding said protein;

(c3) A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in (c 1) and which encodes said protein.

The Arid5a and/or Arid5b genes may also be homologous genes in other species. Such other species include, but are not limited to, mice, rats, rabbits, dogs, monkeys, chimpanzees, apes, cattle, sheep, pigs, horses, sheep, goats, cats, and the like.

The Arid5a and/or Arid5b protein truncations may specifically be (d 1), (d 2), (d 3) or (d 4) as follows:

(d1) A protein shown as 55 th to 148 th amino acid residues in SEQ ID NO.1 of the sequence table;

(d2) A fusion protein obtained by ligating a tag to the amino terminus or the carboxyl terminus of (d 1);

(d3) A protein having any one of the functions (a 1) to (a 3) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of (d 1);

(d4) A protein which is derived from a human and has 98% or more of identity with (d 1) and has any one of functions (a 1) to (a 3).

The labels are specifically shown in table 1.

The Arid5a and/or Arid5b protein truncations may also be homologous proteins in other species having any of functions (a 1) to (a 3). Such other species include, but are not limited to, mice, rats, rabbits, dogs, monkeys, chimpanzees, apes, cattle, sheep, pigs, horses, sheep, goats, cats, and the like.

Any of the above mentioned genes encoding the truncated Arid5a and/or Arid5b protein may specifically be (e 1) or (e 2) or (e 3) as follows:

(e1) DNA molecules encoding the truncated Arid5a and/or Arid5b protein in the DNA molecules shown in SEQ ID No.2 in the sequence table;

(e2) A DNA molecule derived from a human and having more than 95% identity to (e 1) and encoding said Arid5a and/or Arid5b protein truncations;

(e3) A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in (e 1) and which encodes a truncate of said Arid5a and/or Arid5b protein.

The gene encoding the truncated Arid5a protein may also be a homologous gene in other species. Such other species include, but are not limited to, mice, rats, rabbits, dogs, monkeys, chimpanzees, apes, cattle, sheep, pigs, horses, sheep, goats, cats, and the like.

Any of the above biological materials having the Arid5a and/or Arid5b gene may be an expression vector having the Arid5a and/or Arid5b gene. Any of the above biological materials having a gene encoding an Arid5a and/or Arid5b protein truncations may be an expression vector having a gene encoding an Arid5a and/or Arid5b protein truncations. The expression vector is capable of carrying a nucleotide sequence and integrating this sequence into the genome of the cell and is capable of replication in the cell. "expression vectors" include plasmids, cosmids, viruses (phage, animal viruses, plant viruses, etc.) and artificial chromosomes (e.g., YACs). Viruses (also called viral vectors) as expression vectors that are currently possible for clinical gene therapy are as follows: adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, lentiviral vectors, herpes virus vectors, chimeric virus vectors, and other virus vectors.

Any of the above tumors include, but are not limited to, breast cancer.

Any of the above tumor cells include, but are not limited to, breast cancer cells.

The inventor of the present application found that the Arid5a and/or Arid5b proteins have remarkable tumor inhibition function, and can inhibit tumor angiogenesis by selectively stabilizing the expression of anti-angiogenesis gene transcripts (including Endostatin, restin, BAI1, TIMP3, etc.). The inventors of the present application found that the expression of the Arid5a and/or Arid5b genes is inhibited in breast cancer tissues and cells, which may facilitate angiogenesis induced by tumor cells. Overexpression of the Arid5a and/or Arid5b genes can inhibit tumor angiogenesis induced by breast cancer cells, however, tumor angiogenesis can be enhanced by further inhibiting expression of the Arid5a and/or Arid5b genes using shRNA. Consistent with these in vitro observations, overexpression of the Arid5a and/or Arid5b genes in vivo can significantly inhibit tumor growth and metastasis. By analyzing the human tumor tissue sample database, the inventors found that low expression levels of Arid5a and/or Arid5b strongly correlated with poor breast cancer patient survival. Furthermore, the expression level of the Arid5a gene in cancer patient tissues was significantly inversely correlated with the expression level of its target gene Endostatin, restin, BAI 1. These results indicate that Arid5a and/or Arid5b are a potential cancer suppressor gene involved in regulating angiogenic signaling pathways by increasing anti-angiogenic gene expression. Based on the above, the application of the tumor suppressor protein Arid5a and/or Arid5b to improve the clinical tumor treatment effect has very important application prospect.

The application identifies that the human Arid5a and/or Arid5b genes are tumor suppressor genes for the first time, and over-expression of the Arid5a and/or Arid5b genes in tumor cells can significantly suppress tumor angiogenesis and tumor metastasis. The application has important theoretical value for the mechanism research of tumor angiogenesis, tumor growth and metastasis. The application has very important theoretical and practical significance for tumor treatment.

The application is further illustrated below in connection with specific examples, which are not to be construed as limiting the application in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The quantitative tests in the following examples were repeated 3 or more times, and the results were averaged, unless otherwise specified.

pEGFP-N1 vector: clontech Co; the pEGFP-N1 vector expresses EGFP proteins, also known as GFP proteins. MDA-MB-468 cells (human breast cancer cells):HTB-132 ^TM . MDA-MB-231 cells (human breast cancer cells): />HTB-26 ^TM 。

EXAMPLE 1 identification of Arid5a inhibiting tumor-induced angiogenesis New Functions

1. The Arid5a gene (Arid 5a gene is shown as SEQ ID NO.2 of the sequence table, the stop codon is removed from the insertion sequence) is inserted between EcoRI and AgeI cleavage sites of the pEGFP-N1 vector, and the recombinant plasmid pEGFP-N1-Arid5a-GFP is obtained. The recombinant plasmid pEGFP-N1-Arid5a-GFP has been sequenced. In the recombinant plasmid pEGFP-N1-Arid5a-GFP, the inserted DNA molecule and EGFP gene in the vector form fusion gene to express Arid5a-EGFP fusion protein (called A5a-GFP fusion protein for short).

2. The recombinant plasmid pEGFP-N1-Arid5a-GFP was introduced into MDA-MB-468 cells to obtain recombinant cells, which were designated as MDA-MB-468/A5a-GFP cells.

3. The pEGFP-N1 vector was introduced into MDA-MB-468 cells to give recombinant cells, which were designated MDA-MB-468/GFP cells.

4. The recombinant plasmid pEGFP-N1-Arid5a-GFP was introduced into MDA-MB-231 cells to give recombinant cells, which were designated as MDA-MB-231/A5a-GFP cells.

5. The pEGFP-N1 vector was introduced into MDA-MB-231 cells to give recombinant cells, which were designated MDA-MB-231/GFP cells.

6. MDA-MB-468/A5a-GFP cells, MDA-MB-231/A5a-GFP cells, MDA-MB-468/GFP cells and MDA-MB-231/GFP cells were taken, respectively, and the following steps were performed: culturing was performed in parallel, cell lysis was performed after 36 hours, and then lysates were collected for immunoblotting. Results of immunoblotting using the Arid5a antibody are shown in FIG. 1A. Endo-Arid5a represents the cellular endogenous Arid5a protein.

7. MDA-MB-231/GFP cell conditioned medium (labeled MDA-MB-231/Con-CM in the figure) and MDA-MB-231/A5a-GFP cell conditioned medium (labeled MDA-MB-231/Arid5a-CM in the figure) were taken and subjected to the following steps, respectively: culturing under parallel condition, collecting cell culture supernatant after 72 hr, filtering, centrifuging, adding into scratched umbilical vein endothelial cells (HUVECs), culturing for 24 hr, observing endothelial cell migration (as shown in figure 1B left), and calculating scratch area to detect influence of over-expressed Arid5a gene on vascular endothelial cell migration. The same procedure was performed in MDA-MB-468/GFP cells and MDA-MB-468/Arid5a-GFP cells, and the scratch area quantification results are shown on the right side of FIG. 1B.

8. MDA-MB-231/GFP cell conditioned medium (labeled MDA-MB-231/Con-CM in the figure) and MDA-MB-231/A5a-GFP cell conditioned medium (labeled MDA-MB-231/Arid5a-CM in the figure) were taken and subjected to the following steps, respectively: culturing under parallel conditions, collecting cell culture supernatant after 72 hr, filtering, centrifuging, and mixing with HUVECs cells (1×10 ⁴ ) After culturing in a 96-well plate with matrigel, observing the tube forming ability of endothelial cells (as shown in FIG. 1C, left), counting the number of branch points for tube forming to examine the influence of over-expression of Arid5a gene on the tube forming ability of vascular endothelial cells. The same procedure was performed in MDA-MB-468/GFP cells and MDA-MB-468/Arid5a-GFP cells, and the results of the quantification of the tube forming capacity are shown on the right side of FIG. 1C.

9. CD31 immunohistochemical staining was performed on MDA-MB-231/GFP cells and on MDA-MB-231/Arid5a-GFP cell-forming tissues (labeled Con and Arid5a-GFP, respectively, in the figures) to examine the effect of over-expression of the Arid5a gene on angiogenesis in tumor tissues. Typical immunohistochemical staining results are shown on the left of FIG. 1D, and quantitative results for the number of microvessels positive for CD31 staining are shown on the right of FIG. 1D.

10. CD31 immunohistochemical staining was performed on MDA-MB-468/GFP cells and on MDA-MB-468/Arid5a-GFP cell-forming tissues (labeled Con and Arid5a-GFP, respectively, in the figures) to examine the effect of over-expression of the Arid5a gene on angiogenesis in tumor tissues. Typical immunohistochemical staining results are shown on the left of FIG. 1E, and quantitative results for the number of microvessels positive for CD31 staining are shown on the right of FIG. 1E.

The results of fig. 1 show that: both MDA-MB-231/A5a-GFP cells and MDA-MB-468/A5a-GFP cells efficiently expressed the Arid5a-GFP fusion protein (FIG. 1A); overexpression of the Arid5a gene in vitro and in vivo significantly inhibited tumor cell-induced angiogenesis (fig. 1B, 1C, 1D, and 1E).

The above results indicate that: the Arid5a gene is over-expressed to improve the Arid5a protein level, so that angiogenesis induced by tumor cells can be inhibited, namely the Arid5a gene is over-expressed to improve the Arid5a protein level, and the effect of treating tumors is achieved. The above-described functions of the Arid5a protein/Arid 5a gene were found for the first time by the inventors of the present application.

EXAMPLE 2 Arid5a specific stable angiogenesis inhibitor Gene mRNAs

1. Arid5a specifically binds to angiogenesis inhibitor gene mRNAs

In order to further detect the mechanism of inhibiting tumor angiogenesis by Arid5a protein, detection and analysis are carried out on genes related to regulation of tumor angiogenesis.

1. The MDA-MB-231/A5a-GFP cells and MDA-MB-468/A5a-GFP cells of example 1 and their control cells MDA-MB-231/GFP and MDA-MB-468/GFP were taken, and after culturing for 36 hours, total RNAs were extracted and reverse transcribed into cDNAs, respectively, and then RNA-seq detection of angiogenesis-related gene expression was performed, and the results are shown in FIG. 2A.

2. The MDA-MB-231/A5a-GFP cells of example 1 and the control cells MDA-MB-231/GFP cells were cultured for 36 hours, and then total RNAs were extracted and reverse transcribed into cDNAs, followed by PCRarray (QIAGEN) to examine the angiogenesis-related gene expression, and the results are shown in FIG. 2B.

3. MDA-MB-231/A5a-GFP cells and control cells MDA-MB-231/GFP cells thereof are taken and subjected to the following steps: cells were collected at different culture times (0 h, 24h, 36h, 48 h), total RNA was extracted and reverse transcribed into cDNA, and then qPCR was performed to detect the expression amount of the target gene. The results are shown in FIG. 2C.

4. Gene Ontology (Gene ontologiy) analysis is performed on the Gene regulated by Arid5a in the RNA-seq data, and the detection shows that the Gene regulated by Arid5a is significantly enriched in the term of "Angiogenesis" (which indicates that Arid5a regulates Angiogenesis activity, and the result is shown by a red arrow in FIG. 2D.

5. MDA-MB-231/A5a-GFP cells were taken and subjected to the following steps: cell lysis is carried out after 36 hours of culture, and then lysate is collected; GFP antibodies (or isotype IgG) were incubated with Protein A/G Beads (Santa Cruz Co.) for 2h, then the lysates were added and incubation continued for 2h, then centrifuged at 2000rpm at 4℃to thoroughly wash the Beads, and then the total RNA was recovered with Trizol; RT-PCR was performed to detect enrichment of the gene of interest. The results are shown in FIG. 2E. GAPDH served as a negative control and STAT3 served as a positive control.

6. Luciferase reporter assay

The plasmids tested were: recombinant plasmid pEGFP-N1-Arid5a-GFP (designated as Arid5a in the figure) or pEGFP-N1 vector (designated as Control in the figure). The reporter vector constructs were: the schematic diagram of the reporter vector having the 3'UTR of the β -action gene, the reporter vector having the 3' UTR of the COL18A1 gene, the reporter vector having the 3'UTR of the TIMP3 gene, the reporter vector having the 3' UTR of the BAI1 gene, the reporter vector having the 3'UTR of the BAI2 gene, or the reporter vector having the 3' UTR of the TIMP2 gene is shown in FIG. 2F. The test plasmid and reporter vector were co-transfected in HEK293 cells, cell lysis was performed 36 hours after transfection, and lysates were collected and luciferase activity was detected using Dual-LuciferaseReporter Assay System (Promega), as shown in FIG. 2G.

The result of the step one shows that: after overexpression of the Arid5a gene, angiogenesis inhibitor mRNAs were up-regulated and part of angiogenesis promoting genes were down-regulated (FIG. 2A, FIG. 2B, FIG. 2C); moreover, the Arid5a regulated gene is significantly enriched in the term "angiogenesis" (fig. 2D), further indicating its regulatory effect on tumor angiogenesis; the Arid5a protein can target mRNAs that bind to angiogenesis inhibitor genes, but do not bind to angiogenesis promoting genes (FIG. 2E); the Arid5a protein can target the 3'UTR binding to the angiogenesis inhibitor gene (FIG. 2F and FIG. 2G), indicating that Arid5a specifically binds to and promotes expression of the 3' UTR of the angiogenesis inhibitor gene mRNAs.

The results in summary show that: the inventors of the present application have found for the first time a novel function of the Arid5a protein, namely that angiogenesis inhibitor mRNAs can be selectively bound and promoted by targeting binding to 3' UTR, which explains the essential reason why overexpression of the Arid5a gene can prevent tumor angiogenesis.

2. ARID functional region of Arid5a protein stabilizes angiogenesis inhibitor gene mRNAs and tumor angiogenesis

1. MDA-MB-231/A5a-GFP cells (labeled as Arid5a-GFP in the figures) and MDA-MB-231/GFP cells (labeled as Empty Vector in the figures) were taken and the following steps were performed, respectively, to detect half-lives of the angiogenesis inhibitor mRNAs: cells were treated with ActD and DRB (working concentration of ActD 5. Mu.g/mL, working concentration of DRB 5. Mu.g/mL), total RNA was extracted after 0min, 30min, 60min, 90min and 120min, respectively, and then qRT-PCR was performed to detect half-life of the target gene. The results are shown in fig. 3A, 3B, 3C and 3D.

The domain schematic of the Arid5a protein is shown in FIG. 3E. Amino acid residues 55-148 are ARID domains, and the structural analysis is shown in the right side of FIG. 3E.

Several recombinant plasmids were prepared as follows (each recombinant plasmid was sequenced):

inserting a DNA molecule encoding the truncated body 1 in SEQ ID NO.2 of the sequence table into a pEGFP-N1 vector to obtain a recombinant plasmid pEGFP-N1-T1-GFP; the recombinant plasmid pEGFP-N1-T1-GFP expresses the T1-EGFP fusion protein. The truncated body 1 is represented by T1, and is shown as the 1 st to 148 th amino acid residues in SEQ ID NO.1 of the sequence table.

Inserting a DNA molecule encoding the truncated body 2 in SEQ ID NO.2 of the sequence table into a pEGFP-N1 vector to obtain a recombinant plasmid pEGFP-N1-T2-GFP; the recombinant plasmid pEGFP-N1-T2-GFP expresses the T2-EGFP fusion protein. The truncated body 2 is represented by T2, and is shown as amino acid residues 149-594 in SEQ ID NO.1 of the sequence table.

Inserting a DNA molecule encoding the truncated body 3 in SEQ ID NO.2 of the sequence table into a pEGFP-N1 vector to obtain a recombinant plasmid pEGFP-N1-T3-GFP; the recombinant plasmid pEGFP-N1-T3-GFP expresses the T3-EGFP fusion protein. The truncated body 3 is represented by T3, and is shown as 55 th-148 th amino acid residues in SEQ ID NO.1 of the sequence table.

2. Recombinant plasmid pEGFP-N1-T1-GFP was introduced into MDA-MB-231 cells to give recombinant cells, which were designated MDA-MB-231/T1-GFP cells. Recombinant plasmid pEGFP-N1-T2-GFP was introduced into MDA-MB-231 cells to give recombinant cells, which were designated MDA-MB-231/T2-GFP cells. The recombinant plasmid pEGFP-N1-T3-GFP was introduced into MDA-MB-231 cells to give recombinant cells, which were designated MDA-MB-231/T3-GFP cells.

3. The following steps were performed with the MDA-MB-231/A5a-GFP cells (labeled as WT in the figure), the MDA-MB-231/T1-GFP cells (labeled as 1-148 in the figure), the MDA-MB-231/T2-GFP cells (labeled as 149-594 in the figure) and the MDA-MB-231/T3-GFP cells (labeled as 55-148 in the figure) prepared in step 2, respectively, as described below: culturing was performed in parallel, cell lysis was performed after 36 hours, and lysates were collected and immunoblotted with GFP antibody. The results are shown in FIG. 3F.

4. The following steps were performed with the MDA-MB-231/A5a-GFP cells (designated as Arid5a in the figures) and MDA-MB-231/GFP cells (designated as Empty Vector in the figures) of example 1, and with the individual recombinant cells (designated as 1-148, 149-594, 55-148, respectively, according to truncations) prepared in step 2: culturing was performed in parallel, cells were taken after 36 hours, total RNA was extracted and reverse transcribed into cDNA, and then qPCR was performed to detect the expression amount of angiogenesis inhibitor genes. The results are shown in FIG. 3G.

5. Luciferase reporter assay

The test plasmid and reporter vector were co-transfected in HEK293 cells, cell lysis was performed 36 hours after transfection, and lysates were collected and luciferase activity was detected using Dual-LuciferaseReporter Assay System (Promega), as shown in FIG. 3H. The plasmids tested were: recombinant plasmid pEGFP-N1-Arid5a-GFP (labeled Arid5a in the figures) or pEGFP-N1 Vector (labeled Empty Vector in the figures) or recombinant plasmid pEGFP-N1-T1-GFP (labeled 1-148 in the figures) or recombinant plasmid pEGFP-N1-T2-GFP (labeled 149-594 in the figures) or recombinant plasmid pEGFP-N1-T3-GFP (labeled 55-148 in the figures). The reporter vectors are respectively: a reporter vector having a 3' UTR of a β -action gene, a reporter vector having a 3' UTR of a COL18A1 gene, a reporter vector having a 3' UTR of a TIMP3 gene, a reporter vector having a 3' UTR of a BAI1 gene, or a reporter vector having a 3' UTR of a TIMP2 gene.

6. The condition culture supernatant of MDA-MB-231/A5a-GFP cells (labeled A5a CM in the figure) and the condition of MDA-MB-231/GFP cells in example 1 were takenThe following steps were performed, respectively, with the culture supernatant (labeled Con CM in the drawing), the conditioned culture supernatant of MDA-MB-231/T1-GFP cells prepared in step 2 (labeled 1-148CM in the drawing), the conditioned culture supernatant of MDA-MB-231/T2-GFP cells (labeled 149-594CM in the drawing), and the conditioned culture supernatant of MDA-MB-231/T3-GFP cells (labeled 55-148CM in the drawing): under parallel conditions with HUVECs cells (1X 10 ⁴ ) Endothelial cells were tested for their ability to form tubes after 12 hours of incubation. The results are shown in FIG. 3I.

The result of the second step shows that: after overexpression of the T1 or T3 truncate genes, the angiogenesis inhibiting gene mRNAs are up-regulated (fig. 3G); t1 truncations or T3 truncations may target 3' UTRs that bind angiogenesis inhibiting genes to stabilize their mRNAs (FIG. 3H); overexpression of the T1 or T3 truncate gene inhibited tumor-induced angiogenesis (FIG. 3I).

The above results indicate that: the ARID functional region of the Arid5a protein plays an important role in specifically stabilizing angiogenesis inhibitor genes and inhibiting tumor angiogenesis.

EXAMPLE 3 overexpression of the Arid5a Gene (increasing Arid5a protein levels) inhibiting tumor growth and metastasis

1. The following steps were performed with the MDA-MB-231/A5a-GFP cells (labeled as Arid5a-GFP in the figure) and MDA-MB-468/GFP cells (labeled as Control Vector in the figure) in example 1, respectively: baLB/c nude mice injected subcutaneously on the back (1X 10 per mouse injection) ⁸ Individual cells/100 μl PBS buffer), days from the start of injection. In situ tumor volumes were measured daily on days 14 to 36, as shown in fig. 4A (average of 5 mice) for changes over time, and photographs of in situ tumors of mice after 36 days of injection are shown in fig. 4B. Mice were sacrificed and dissected on day 36, and all mice developed lung metastases except for in situ tumors. The lungs were taken and photographed, see left image of fig. 4C. The number of white nodules in the lungs was counted, see right panel of fig. 4C (average of 5 mice).

2. MDA-MB-231 cells were taken and injected subcutaneously back into BALB/c nude mice (1X 10 per mouse injection) ⁸ Individual cells/100 μl PBS buffer). Days were counted from the injection of MDA-MB-231 cells. 4 thIn situ tumor average diameter of each mouse at day 8>5mm. Recombinant adenovirus was injected every other day (1×10 per mouse injection) starting on day 48 ¹⁰ pfu), 5 times total injection. Two treatment groups were set, and recombinant adenovirus expressing the Arid5a gene (Arid 5a gene is shown as SEQ ID No.2 of the sequence Listing, recombinant adenovirus expressing the Arid5a gene is represented by Arid5a adenovrus or Ad-Arid5 a) or control adenovirus (represented by Control adenovirus or Ad-Con) was administered separately, and the control adenovirus was different from the recombinant adenovirus expressing the Arid5a gene only in that it did not have the Arid5a gene. In situ tumor volumes were measured daily on days 24 to 64 and the change in situ tumor volume over time is shown in fig. 4A (average of 5 mice). On day 64, mice were sacrificed and tumor masses were taken to compare tumor sizes, see fig. 4B. Expression of the Arid5a-GFP fusion protein in tumor tissues was examined by immunoblotting and the results are shown in FIG. 4C (wherein C1, C2 represent tumors treated with Ad-Con expressing GFP; T1, T2 represent tumors treated with Ad-Arid5 a). The lung was taken for pathological section and HE stained, see left fig. 4D. The number of metastases was counted according to the results of HE staining, see figure 4D right (average of 5 mice). CD31 immunohistochemical staining results were shown on the left of FIG. 4E and quantitative results on the number of microvessels positive for CD31 staining were shown on the right of FIG. 4E using tumor tissues treated with Ad-Control and Ad-Arid5a (labeled Ad-Control and Ad-Arid5a, respectively).

As a result, arid5a was found to significantly inhibit tumor growth and tumor cell lung metastasis and tumor angiogenesis in vivo. In conclusion, the experimental result shows that the over-expression of the Arid5a gene (improving the Arid5a protein level) can obviously inhibit the growth, the metastasis and the tumor angiogenesis of tumors.

Any numerical value recited in this disclosure includes all values incremented by one unit from the lowest value to the highest value if there is only a two unit interval between any lowest value and any highest value. For example, if the amount of one component, or the value of a process variable such as temperature, pressure, time, etc., is stated to be 50-90, it is meant in this specification that values such as 51-89, 52-88 … …, and 69-71, and 70-71 are specifically recited. For non-integer values, 0.1, 0.01, 0.001 or 0.0001 units may be considered as appropriate. This is only a few examples of the specific designations. In a similar manner, all possible combinations of values between the lowest value and the highest value enumerated are to be considered to be disclosed.

It should be noted that the above-described embodiments are only for explaining the present application and do not constitute any limitation of the present application. The application has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the application as defined in the appended claims, and the application may be modified without departing from the scope and spirit of the application. Although the application is described herein with reference to particular means, materials and embodiments, the application is not intended to be limited to the particulars disclosed herein, as the application extends to all other means and applications which perform the same function.

Sequence listing

<110> national academy of medical science microcirculation institute

<120> tumor angiogenesis regulatory protein and application thereof

<130> 1

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1133

<212> PRT

<213> Arid5a

<400> 1

Met Pro Val Gln Ala Pro Gln Trp Thr Asp Phe Leu Ser Cys Pro Ile

1 5 10 15

Cys Thr Gln Thr Phe Asp Glu Thr Ile Arg Lys Pro Ile Ser Leu Gly

20 25 30

Cys Gly His Thr Val Cys Lys Met Cys Leu Asn Lys Leu His Arg Lys

35 40 45

Ala Cys Pro Phe Asp Gln Thr Thr Ile Asn Thr Asp Ile Glu Leu Leu

50 55 60

Pro Val Asn Ser Ala Leu Leu Gln Leu Val Gly Ala Gln Val Pro Glu

65 70 75 80

Gln Gln Pro Ile Thr Leu Cys Ser Gly Val Glu Asp Thr Lys His Tyr

85 90 95

Glu Glu Ala Lys Lys Cys Val Glu Glu Leu Ala Leu Tyr Leu Lys Pro

100 105 110

Leu Ser Ser Ala Arg Gly Val Gly Leu Asn Ser Thr Thr Gln Ser Val

115 120 125

Leu Ser Arg Pro Met Gln Arg Lys Leu Val Thr Leu Val His Cys Gln

130 135 140

Leu Val Glu Glu Glu Gly Arg Ile Arg Ala Met Arg Ala Ala Arg Ser

145 150 155 160

Leu Gly Glu Arg Thr Val Thr Glu Leu Ile Leu Gln His Gln Asn Pro

165 170 175

Gln Gln Leu Ser Ser Asn Leu Trp Ala Ala Val Arg Ala Arg Gly Cys

180 185 190

Gln Phe Leu Gly Pro Ala Met Gln Glu Glu Ala Leu Lys Leu Val Leu

195 200 205

Leu Ala Leu Glu Asp Gly Ser Ala Leu Ser Arg Lys Val Leu Val Leu

210 215 220

Phe Val Val Gln Arg Leu Glu Pro Arg Phe Pro Gln Ala Ser Lys Thr

225 230 235 240

Ser Ile Gly His Val Val Gln Leu Leu Tyr Arg Ala Ser Cys Phe Lys

245 250 255

Val Thr Lys Arg Asp Glu Asp Ser Ser Leu Met Gln Leu Lys Glu Glu

260 265 270

Phe Arg Thr Tyr Glu Ala Leu Arg Arg Glu His Asp Ser Gln Ile Val

275 280 285

Gln Ile Ala Met Glu Ala Gly Leu Arg Ile Ala Pro Asp Gln Trp Ser

290 295 300

Ser Leu Leu Tyr Gly Asp Gln Ser His Lys Ser His Met Gln Ser Ile

305 310 315 320

Ile Asp Lys Leu Gln Thr Pro Ala Ser Phe Ala Gln Ser Val Gln Glu

325 330 335

Leu Thr Ile Ala Leu Gln Arg Thr Gly Asp Pro Ala Asn Leu Asn Arg

340 345 350

Leu Arg Pro His Leu Glu Leu Leu Ala Asn Ile Asp Pro Ser Pro Asp

355 360 365

Ala Pro Pro Pro Thr Trp Glu Gln Leu Glu Asn Gly Leu Val Ala Val

370 375 380

Arg Thr Val Val His Gly Leu Val Asp Tyr Ile Gln Asn His Ser Lys

385 390 395 400

Lys Gly Ala Asp Gln Gln Gln Pro Pro Gln His Ser Lys Tyr Lys Thr

405 410 415

Tyr Met Cys Arg Asp Met Lys Gln Arg Gly Gly Cys Pro Arg Gly Ala

420 425 430

Ser Cys Thr Phe Ala His Ser Gln Glu Glu Leu Glu Lys Phe Arg Lys

435 440 445

Met Asn Lys Arg Leu Val Pro Arg Arg Pro Leu Ser Ala Ser Leu Gly

450 455 460

Gln Leu Asn Glu Val Gly Leu Pro Ser Ala Ala Ile Leu Pro Asp Glu

465 470 475 480

Gly Ala Val Asp Leu Pro Ser Arg Lys Pro Pro Ala Leu Pro Asn Gly

485 490 495

Ile Val Ser Thr Gly Asn Thr Val Thr Gln Leu Ile Pro Arg Gly Thr

500 505 510

Asp Pro Ser Tyr Asp Ser Ser Leu Lys Pro Gly Lys Ile Asp His Leu

515 520 525

Ser Ser Ser Ala Pro Gly Ser Pro Pro Asp Leu Leu Glu Ser Val Pro

530 535 540

Lys Ser Ile Ser Ala Leu Pro Val Asn Pro His Ser Ile Pro Pro Arg

545 550 555 560

Gly Pro Ala Asp Leu Pro Pro Met Pro Val Thr Lys Pro Leu Gln Met

565 570 575

Val Pro Arg Gly Ser Gln Leu Tyr Pro Ala Gln Gln Thr Asp Val Tyr

580 585 590

Tyr Gln Asp Pro Arg Gly Ala Ala Pro Pro Phe Glu Pro Ala Pro Tyr

595 600 605

Gln Gln Gly Met Tyr Tyr Thr Pro Pro Pro Gln Cys Val Ser Arg Phe

610 615 620

Val Arg Pro Pro Pro Ser Ala Pro Glu Pro Ala Pro Pro Tyr Leu Asp

625 630 635 640

His Tyr Pro Pro Tyr Leu Gln Glu Arg Val Val Asn Ser Gln Tyr Gly

645 650 655

Thr Gln Pro Gln Gln Tyr Pro Pro Ile Tyr Pro Ser His Tyr Asp Gly

660 665 670

Arg Arg Val Tyr Pro Ala Pro Ser Tyr Thr Arg Glu Glu Ile Phe Arg

675 680 685

Glu Ser Pro Ile Pro Ile Glu Ile Pro Pro Ala Ala Val Pro Ser Tyr

690 695 700

Val Pro Glu Ser Arg Glu Arg Tyr Gln Gln Ile Glu Ser Tyr Tyr Pro

705 710 715 720

Val Ala Pro His Pro Thr Gln Ile Arg Pro Ser Tyr Leu Arg Glu Pro

725 730 735

Pro Tyr Ser Arg Leu Pro Pro Pro Pro Gln Pro His Pro Ser Leu Asp

740 745 750

Glu Leu His Arg Arg Arg Lys Glu Ile Met Ala Gln Leu Glu Glu Arg

755 760 765

Lys Val Ile Ser Pro Pro Pro Phe Ala Pro Ser Pro Thr Leu Pro Pro

770 775 780

Thr Phe His Pro Glu Glu Phe Leu Asp Glu Asp Leu Lys Val Ala Gly

785 790 795 800

Lys Tyr Lys Gly Asn Asp Tyr Ser Gln Tyr Ser Pro Trp Ser Cys Asp

805 810 815

Thr Ile Gly Ser Tyr Ile Gly Thr Lys Asp Ala Lys Pro Lys Asp Val

820 825 830

Val Ala Ala Gly Ser Val Glu Met Met Asn Val Glu Ser Lys Gly Met

835 840 845

Arg Asp Gln Arg Leu Asp Leu Gln Arg Arg Ala Ala Glu Thr Ser Asp

850 855 860

Asp Asp Leu Ile Pro Phe Gly Asp Arg Pro Thr Val Ser Arg Phe Gly

865 870 875 880

Ala Ile Ser Arg Thr Ser Lys Thr Ile Tyr Gln Gly Ala Gly Pro Met

885 890 895

Gln Ala Met Ala Pro Gln Gly Ala Pro Thr Lys Ser Ile Asn Ile Ser

900 905 910

Asp Tyr Ser Pro Tyr Gly Thr His Gly Gly Trp Gly Ala Ser Pro Tyr

915 920 925

Ser Pro His Gln Asn Ile Pro Ser Gln Gly His Phe Ser Glu Arg Glu

930 935 940

Arg Ile Ser Met Ser Glu Val Ala Ser His Gly Lys Pro Leu Pro Ser

945 950 955 960

Ala Glu Arg Glu Gln Leu Arg Leu Glu Leu Gln Gln Leu Asn His Gln

965 970 975

Ile Ser Gln Gln Thr Gln Leu Arg Gly Leu Glu Ala Val Ser Asn Arg

980 985 990

Leu Val Leu Gln Arg Glu Ala Asn Thr Leu Ala Gly Gln Ser Gln Pro

995 1000 1005

Pro Pro Pro Pro Pro Pro Lys Trp Pro Gly Met Ile Ser Ser Glu Gln

1010 1015 1020

Leu Ser Leu Glu Leu His Gln Val Glu Arg Glu Ile Gly Lys Arg Thr

1025 1030 1035 1040

Arg Glu Leu Ser Met Glu Asn Gln Cys Ser Leu Asp Met Lys Ser Lys

1045 1050 1055

Leu Asn Thr Ser Lys Gln Ala Glu Asn Gly Gln Pro Glu Pro Gln Asn

1060 1065 1070

Lys Val Pro Ala Glu Asp Leu Thr Leu Thr Phe Ser Asp Val Pro Asn

1075 1080 1085

Gly Ser Ala Leu Thr Gln Glu Asn Ile Ser Leu Leu Ser Asn Lys Thr

1090 1095 1100

Ser Ser Leu Asn Leu Ser Glu Asp Pro Glu Gly Gly Gly Asp Asn Asn

1105 1110 1115 1120

Asp Ser Gln Arg Ser Gly Val Thr Pro Ser Ser Ala Pro

1125 1130

<210> 2

<211> 3402

<212> DNA

<213> Arid5a

<400> 2

atgcctgtac aagctccaca atggacggat ttcctctcct gcccaatttg cactcagact 60

ttcgacgaaa caattcgaaa gcccatcagt ttgggttgtg gccatactgt ctgcaagatg 120

tgcctgaata aactccaccg caaggcttgc ccatttgacc agaccactat caatacagac 180

attgagctcc tccctgtgaa ctcagcattg ctgcagctcg tgggtgctca ggtccctgag 240

cagcagccta ttactttgtg tagtggggtt gaagacacaa agcattatga ggaagccaag 300

aaatgtgtag aagaattagc attgtactta aaaccgctca gcagtgctag aggagtgggt 360

ctgaacagca ctactcagag tgttctgagt cgcccaatgc agaggaagct tgtgactcta 420

gtccactgtc aactagtaga agaagaaggc aggattcgtg ccatgagggc agctcgatct 480

ttaggtgaac gaacagttac agagctcatt ctccagcacc agaatcctca gcaactctct 540

tccaatcttt gggcagcagt aagggctagg ggatgccagt tccttggacc agcaatgcag 600

gaggaagctt taaagttggt tctgctggcc ttagaagatg gttctgcttt gtcaagaaaa 660

gtattggttc tgtttgtggt gcaaagattg gagccacggt ttcctcaagc ctctaaaact 720

agcattgggc atgttgttca gctcctttat agagcctcct gtttcaaggt caccaaacgt 780

gatgaagact cttctttgat gcagctgaaa gaagaattta gaacctatga agctctgcgg 840

cgagaacatg actcccagat agtgcagatt gctatggaag caggcttacg aattgcaccg 900

gaccagtggt cctctttgct ttatggagac cagtctcaca aatctcatat gcagtccatt 960

attgacaagt tgcagactcc agcctctttt gcacagagtg ttcaggaact aacaattgct 1020

ctccagcgaa ctggagaccc agcaaacttg aaccgactaa gaccccattt ggagctgtta 1080

gcaaacattg accctagtcc agatgctcct cctcctactt gggaacagct ggaaaatggg 1140

ctggttgctg tgcgtacagt ggtacatggg ctggttgatt atatccagaa ccacagcaaa 1200

aaaggagcag atcagcagca gcctccacag catagcaaat acaaaacata catgtgtcga 1260

gatatgaagc agagaggagg atgccctcgt ggggccagct gtacatttgc acactcacag 1320

gaggaactgg aaaaatttcg taaaatgaat aagcgcctgg ttccgagaag acccttgagt 1380

gcctctttgg gtcaacttaa tgaggtgggc ctgccttcag cagctatcct tccagatgaa 1440

ggtgcagtgg atctccctag cagaaaacct cctgctctgc caaatggaat tgtatcaaca 1500

gggaatacag taacacagct tattccgcga gggacagacc ccagctatga ttctagtctg 1560

aaaccaggaa aaatagatca tctgagcagt agtgctcctg gatcccctcc tgacctgcta 1620

gaatctgttc ctaagagtat ttctgcctta cctgtgaatc cacattctat acctccaagg 1680

gggccagcag atctgcctcc aatgcctgtt accaaaccac ttcagatggt acctcgaggt 1740

tctcagttat atccagcaca acagacggat gtttattatc aggatcctcg aggagcagct 1800

ccgccatttg aaccagcacc ttatcagcag ggtatgtatt atactccacc accacaatgt 1860

gtgtcccgct ttgtccgacc tccaccatct gctcctgaac ctgctcctcc ctacttggat 1920

cattatccac cctacctcca agaacgtgtt gtaaactctc agtatggcac acagccacag 1980

cagtacccac ctatataccc atctcactat gatggccgtc gagtgtaccc tgctccgtct 2040

tacacaagag aagagatatt ccgagaaagc cctataccca ttgagattcc acctgcagca 2100

gtaccatcgt atgtaccaga atccagagaa agataccaac agatcgagag ttactatcca 2160

gtggctcctc atccaactca gatcagacct tcgtacctca gagaacctcc ttatagccgg 2220

cttcctcctc ctccccagcc tcatcctagt ctagatgaac tacatcgccg acgaaaggaa 2280

ataatggccc agctagagga aagaaaggtt atctctccac ctccttttgc accttcacca 2340

accttgcctc ctacctttca tccggaagaa tttttggatg aagacttgaa ggtagctggg 2400

aaatacaaag gaaatgatta tagccaatac tctccctggt catgtgacac catcggctcc 2460

tacattggaa ccaaagatgc aaaacccaaa gatgttgtgg cagcagggag tgtggaaatg 2520

atgaatgtgg agagtaaagg aatgagggac cagcgattag atcttcagag aagagcagca 2580

gaaaccagtg atgatgacct catcccattt ggagaccgac caacagtgtc tcggtttggt 2640

gccatctcac gaacttccaa aactatatat cagggtgctg gtccaatgca ggctatggca 2700

cctcagggag ctcctacaaa atctattaac atttcagatt atagtccata tggaacccac 2760

ggtggctggg gagcttctcc atattcacct catcaaaaca taccttctca gggacacttc 2820

agtgagaggg agagaatatc tatgtcagaa gtggccagtc atggaaaacc ccttccatct 2880

gctgagagag aacagctacg actagaattg cagcaattga accatcagat tagccagcag 2940

acccagctac gtggactaga ggctgttagt aacaggctgg tgttgcagag ggaggcaaac 3000

accctggcag gccagtcaca gcccccacca ccgccacctc caaaatggcc tgggatgatc 3060

tcaagtgagc agttgagctt ggaactgcac caggtggaaa gggaaatcgg gaagagaaca 3120

cgggaactga gtatggaaaa ccagtgttct ctggacatga aaagcaaact gaatacaagt 3180

aaacaagcag aaaatggaca accagaacca caaaacaagg ttccggctga ggaccttaca 3240

ttgacattca gtgatgtacc aaatggatca gccttgacac aagagaatat cagcctccta 3300

tcaaacaaga ccagctctct gaacctgtca gaggaccctg agggaggagg ggataataat 3360

gactcccaga gatcaggagt tactcccagt tctgctcctt aa 3402

Claims

1. A protein truncate, characterized in that the protein truncate is (d 1) or (d 2): (d1) A protein shown as 55 th to 148 th amino acid residues in SEQ ID NO.1 of the sequence table; (d2) A fusion protein obtained by ligating a tag to the amino terminus or the carboxyl terminus of (d 1).

2. A gene encoding the protein truncate of claim 1.

3. The use of a protein truncate according to claim 1 in the preparation of a medicament for the treatment of a tumor; the functions of the medicine are as follows (a 1) and/or (a 2) and/or (a 3) and/or (a 4): (a 1) treating a tumor; (a 2) inhibiting tumor growth; (a 3) inhibiting tumor metastasis; (a 4) inhibiting tumor angiogenesis.

4. The biomaterial produced by the gene of claim 2.

5. Use of the biomaterial prepared from the gene of claim 2 in the preparation of a medicament for treating tumor; the functions of the medicine are as follows (a 1) and/or (a 2) and/or (a 3) and/or (a 4): (a 1) treating a tumor; (a 2) inhibiting tumor growth; (a 3) inhibiting tumor metastasis; (a 4) inhibiting tumor angiogenesis.